Many cell-based and DNA-based analytical methods require releasing DNA from inside the cell to facilitate analysis. Opening the cells to release the DNA is called ‘lysis.’ For example, methods used to investigate the microbiome using DNA sequencing techniques first require lysis of microbes so the DNA can be extracted. Most microbiomes are communities of bacteria, archaea and fungi that vary tremendously in their susceptibility to lysis techniques. Differential susceptibility presents a significant problem to researchers, who want to ensure that the toughest (usually Gram-positive) and the easiest (usually Gram-negative) to lyse bacteria are represented in proportion to their population in the original sample. Unfortunately, most microbial lysis protocols work well for some microbes, but poorly for others. Additionally, rapid and simple alkaline lysis techniques used to recover plasmid DNA typically also remove the microbial genomic DNA, which is the target for microbiome screening (Alkaline Lysis opens cells but removes gDNA—Birnboim, H. C. and Doly, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res. 7(6), 1979, 1513-1524; KOH lysis recovers bacterial genomic DNA—Raghunathan, Arumugham et al. “Genomic DNA Amplification from a Single Bacterium.” Applied and Environmental Microbiology 71.6 (2005): 3342-3347. PMC. Web. 29 Sept. 2016). There are multiple lysis techniques known in the art that attack cellular integrity based on different biochemical methods, including lysozyme (enzymatic attack on the peptidoglycan cell wall), strong base (chemical attack), detergent (solubilizes cell membranes), bead beating or shaking (mechanical disruption), and heat (Comparison of lysis techniques for microbiome—Sanqing Yuan, Dora B. Cohen, Jacques Ravel, Zaid Abdo, Larry J. Forney. Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome. PLoS ONE 7(3): e33865. doi: 10.1371/journal.pone.0033865; DNA extraction methods affect microbiome profiling results: Wagner Mackenzie B, Waite D W, Taylor M W. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences. Frontiers in Microbiology. 2015;6:130. doi:10.3389/fmicb.2015.00130). Most published or commercially available DNA preparation methods use one or more of these methods to lyse cells, usually in sequential steps that can take a significant amount of time, especially when handling many samples at once. While individual lysis methods are usually sufficient for applications where incomplete or partial lysis yields sufficient DNA for the protocol being performed, they often do not yield DNA from microbiome samples in proportion to the original community, and may fail to lyse certain microbes altogether. For example, a detergent-based lysis may disrupt a subset of cells with weak cell walls and strong cell membranes, but not open detergent-resistant microbes with strong cell walls, leading to under-representation or absence of DNA from detergent resistant cells in the resulting DNA preparation. In another example, bead beating of microbes sufficient to lyse cells with strong cell membranes may shear or destroy DNA released early in the process from easily lysed cells. Additionally, the various methods of lysis tend to be incompatible with each other, and need to be performed sequentially if used in combination. For example, lysozyme will not work in the presence of detergents or strong base. Certain detergents precipitate in the presence of strong base. Bead beating is difficult to combine with a heating process. While individual shortcomings may be overcome by running separate lysis protocols in series, this increases the complexity, time, and cost involved. Importantly, detergents such as sodium dodecyl sulfate (SDS) must be removed after lysis, because SDS interferes with downstream DNA manipulation. Additionally, certain microbes may be resistant to lysis protocols run sequentially, depending on protocol order. For example, certain microbes with tough peptidoglycan cell walls may have an outer envelope of lipid bi-layer that protects from an initial treatment with strong base or lysozyme. Only a simultaneous combination of multiple methods may be effective, or a long sequence of multiple steps, to yield DNA from all microbes in a sample.
The methods disclosed herein streamline lysis for applications and techniques where proportional lysis is desired or necessary, such as microbiome research, by combining multiple lysis methods into a simple, rapid protocol that yields a more representative DNA profile across a sample containing different cellular constituents, such as the microbiome.